Neurosteroids, steroids synthesized de novo in the brain, have been implicated in functions ranging from stress, depression, anxiety, to cognition. One neurosteroid is progesterone, a classic sex hormone involved in the regulation of reproduction. We have previously shown that peripheral estradiol (E2) increases hypothalamic neuroprogesterone (neuroP) synthesis, which initiates the luteinizing hormone (LH) surge. The LH surge triggers ovulation and the luteinization of the ruptured follicle - critical events in reproduction. Within the hypothalamus, astrocytes respond to E2 stimulation by increasing neuroP synthesis. Preliminary data point to E2-signaling that requires an interaction between membrane-associated estrogen receptor-1 (mER1) and a group 1 metabotropic glutamate receptor (mGluR1a). The resulting rise in free cytoplasmic calcium is sufficient to stimulate neuroP synthesis. Since GnRH neurons that control the LH surge do not have steroid receptors, other neurons are required to transmit steroid information to them. Kisspeptin neurons have been suggested as these intervening neurons: they project to GnRH neurons, potently excite them, and express ER1 and progesterone receptor (PR). Based on these results, we propose to test the hypothesis that estrogen positive feedback requires E2-induced neuroP synthesis involving a mER1 and mGluR1a interaction, and the activation of kisspeptin neurons. Three experiments are proposed to study E2 signaling in vitro and in vivo. Three subhypotheses will be tested: 1) E2-induced neuroP synthesis requires activation of the mGluR; 2) E2 stimulates the synthesis of neuroP by activating StAR; and 3) estrogen positive feedback requires PR-mediated activation of periventricular kisspeptin neurons. These studies will use astrocyte cultures to work out cell signaling pathways, and whole animal experiments to verify the in vitro results and tie them to regulation of the LH surge. The proposed studies will test a novel mechanism regulating the transduction of E2 information into neural signals that stimulate the release of GnRH resulting in the LH surge. PUBLIC HEALTH RELEVANCE The proposed studies at the basic level seek to provide an integrated model of estrogen positive feedback to understand the mechanisms that control hypothalamic regulation of the luteinizing hormone (LH) surge and ovulation which are important for both regulating fertility as well as treating infertility. Approximately 12% of women (7.3 million) in the United States aged 15-44 had fertility difficulties (National Center for Health Statistics of the Centers for Disease Control and Prevention) because of ovulatory dysfunction that is controlled by the LH surge. This type of infertility exhibits excess estrogen production without ovulation that can lead to polycystic ovary syndrome, irregular vaginal bleeding, metabolic abnormalities as well as endometrial polyps, hyperplasia, and even cancer.